CAREER: Establishment of Replication Timing and Patterning in Mammalian Cells

智力优势：染色质的结构，即真核细胞核中发现的基因组DNA和蛋白质的复合体，控制着细胞过程，包括DNA复制、转录和转录后基因调控。但是，如何实现这一点是一个有待明确阐明的重要问题。在哺乳动物细胞中，染色体结构域占据特定的核位置，并在细胞分裂周期的S阶段的特定时间进行复制。在给定的细胞类型中，DNA复制的时空模式是可遗传的，并在细胞周期的G1期建立。然而，人们对支配DNA复制时空模式的事件和分子知之甚少。起源识别复合体(Origin Recognition Complex，ORC)在DNA复制的启动、染色质的组织和细胞周期进程中起着至关重要的作用。人ORC的最大亚基ORC1与染色质的结合是G1期间前RC组装的必需步骤。类似地，Orc2-5以细胞周期依赖的方式显示与染色质的动态关联。ORC和控制复制时机和构图的染色质结构之间的相互作用仍有待阐明。本项目的目标是确定ORC如何在G1期间对表观遗传标记做出贡献和/或读取表观遗传标记。该项目将解决ORC在建立空间图案和G1复制时机方面的作用，并确定染色质结构在调节ORC与染色质结合中的作用。这项研究在将分子生物学和生物化学与最先进的活细胞成像技术相结合方面具有创新性。这个项目意义重大，因为它旨在促进我们对染色质组织和复制是如何在活细胞中协调的理解。广泛的影响：这项研究将具有强大而积极的推广潜力，因为它使用了广泛的高分辨率显微镜以及延时活细胞成像，这不仅对于理解关于复制的关键生物学问题至关重要，而且也是本科生欣赏生物学的平台。这种方法将在几个方面得到应用，包括教授本科生，为伊利诺伊大学和社区学院的学生提供研究机会，指导未来的研究人员，以及为学生组织一次研讨会，让他们欣赏基础研究的兴奋。这些活动都将涉及妇女和少数族裔学生。这个项目综合了生物化学、细胞生物学和分子生物学的应用，以吸引来自不同背景的学生的兴趣，包括生物学、物理、化学和生物工程。用少量的时间投入，让年轻而聪明的学生在生命的早期进行研究将是非常有益的，并向他们灌输科学意识。

Intellectual merit:The structure of chromatin, the complex of genomic DNA and proteins found in the nucleus of eukaryotic cells, governs cellular processes including DNA replication, transcription and post-transcriptional gene regulation. But, how that is accomplished is an important question that remains to be clearly elucidated. In mammalian cells, chromosomal domains occupy a specific nuclear position and replicate at defined times during S-phase of the cell division cycle. The spatiotemporal pattern of DNA replication within a given cell type is heritable and is established during the G1 phase of the cell cycle. There is, however, minimal understanding of the events and molecules that govern the spatiotemporal patterning of DNA replication. The Origin Recognition Complex (ORC) plays critical roles in the initiation of DNA replication, chromatin organization and cell cycle progression. The binding of the largest subunit of human ORC, Orc1 to chromatin is an obligatory step for pre-RC assembly during G1. Similarly, Orc2-5 display dynamic association with chromatin in a cell cycle-dependent manner. The interplay between ORC and chromatin structure that govern replication timing and patterning remain to be elucidated. The objective of the present project is to determine how ORC contributes to and/or reads epigenetic marks during G1. The project will address the role of ORC in establishing spatial patterning and timing of replication in G1 and determine the role of chromatin architecture in modulating ORC binding to chromatin. This research is innovative in its combination of molecular biology and biochemistry with state of the art live cell imaging. This project is highly significant as it aims to advance our understanding of how chromatin organization and replication are coordinated in living cells.Broader impacts:This research will have a strong and active outreach potential, since it utilizes extensive high-resolution microscopy as well as time-lapse live cell imaging that is crucial not only to understand key biological questions on replication, but also to serve as a platform for undergraduate students to appreciate biology. This approach will be applied in several ways including by teaching undergraduate students, by providing research opportunities to University of Illinois and community college students, by mentoring future researchers, and by organizing a symposium for students to appreciate the excitement of basic research. These activities will all involve women and minority students. This project integrates the use of Biochemistry, Cell Biology and Molecular Biology to capture the interest of students from varied backgrounds, including Biology, Physics, Chemistry and Bioengineering. With a small investment of time, exposing young and bright students to research early on in life will be highly beneficial and instill in them a sense of scientific awareness.